This invention relates to targeted gene delivery. More particularly, the invention relates to a composition comprising a conjugate of poly(ethylene glycol)-grafted cationic polymers, i.e. polyamines, and a cell targeting molecule for gene delivery to the target cells.
Gene therapy has represented a new paradigm for therapy of human disease and for drug delivery. The implicit emphasis of prior research has been on determining the safety of gene transfer procedures, often placing efficacy as a secondary goal. A major technical impediment to gene transfer is the lack of an ideal gene delivery system. If it were possible to deliver the gene to the appropriate specific cells in sufficient quantities without adverse side effects, gene therapy would be efficacious. Currently very few organs or cells can be specifically targeted for gene delivery.
There are many established protocols for transferring genes into cells, including calcium phosphate precipitation, electroporation, particle bombardment, liposomal delivery and viral-vector delivery. Although all of these methods can be used for mammalian cultured cells, there are many difficulties in introducing genes into target cells in vivo.
Transfection methods using retroviral or adenoviral vectors overcome some of these limitations. Retroviral vectors, in particular, have been used successfully for introducing exogenous genes into the genomes of actively dividing cells such that stable transformants are obtained. D. G. Miller et al., Gene Transfer by Retrovirus Vectors Occurs Only in Cells that are Actively Replicating at the Time of Infection. 10 Mol. Cell Biol. 4239-4242 (1990). Viral vector systems often involve complementation of defective vectors by genes inserted into xe2x80x98helperxe2x80x99 cell lines to generate the transducing infectious agent. However, it is well known that the host immune response to adenoviruses limits their use as a transfer facilitating agent to a single administration. To address this limitation fusion peptides of the influenza virus hemagglutinin have been employed to replace adenoviruses as endosomal lytic agents, but they have met with limited success. S. Gottschalk et al., A Novel DNA-Peptide Complex for Efficient Gene Transfer and Expression in Mammalian Cells, 3 Gene Ther. 448-457 (1996). However, despite their high transfection efficiency in vitro, inserting genes into the host cell""s genome in this method depends on the viral infection pathway. Application of the viral infection pathway for human gene therapy introduces serious concerns about endogenous virus recombination, oncogenic effects, and inflammatory or immunologic reactions. G Ross et al., Gene Therapy in the United States: A Five-Year Status Report. 7 Hum. Gene Ther., 1781-1790 (1996). Because of these concerns, the use of viral vectors for human gene therapy has been extremely limited.
On the other hand, non-viral gene delivery systems such as cationic liposomes or synthetic cationic polymers, e.g.poly-L-lysine (PLL) and polyamines, are being widely sought as alternatives. M. A. Wolfert et al., Characterization of Vectors for Gene Therapy Formed by Self-Assembly of DNA with Synthetic Block Co-Polymers. 7 Hum. Gene. Ther., 2123-2133 (1996); A V Kabanov and V A Kabanov DNA Complexes with Polycations for the Delivery of Genetic Materials into Cells. 6 Bioconj. Chem., 7-20 (1995). There are several advantages to the use of non-viral based gene therapies including their relative safety and low cost of manufacture. The major limitation of plasmid DNA(pDNA)-based approaches has been that both the efficiency of gene delivery to important somatic targets (i.e., liver, lung and tumors) and in vivo gene expression levels are lower using non-viral approaches than those using viral vectors. Polyethylenimine (PEI), one of the most commonly used cationic polymers, mediates a high degree of transfection due to the release of pDNA from the endosome to the cytosol. O.Boussif, F. Lezoualc""h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix, J. P. Behr, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. USA. 92 (1995) 7297-7301. Although PEI condenses pDNA into complexes of less than 50 nm in salt-free buffer, these complexes aggregate immediately under physiological conditions such as in salt or bovine serum albumin.
Receptor-mediated gene delivery has its advantages and limitations. Its advantages for use in gene therapy are as follows. First, the gene delivery carrier can be designed and customized for a specific target receptor. Second, the DNA does not have to integrate into the host cell genome to be expressed. Third, the delivery system is theoretically not limited by the size of the transgene. Finally, the technique does not involve the use of potentially infectious agents. There are also disadvantages that must be overcome before this procedure can be routinely used for human gene therapy. For example, the transgene is not integrated into the host cell chromosomes, or its expression is transient. Therefore, it will most likely be necessary to subject patients to multiple injections of a gene of interest. The DNA-targeting moiety(TM) complexes are difficult to prepare and, until recently, little was known about their structure-function relationship. Also, there is only a fragmentary understanding of the biological process involved in the transfer of the transgene into the cell and its subsequent expression. These and other features of this system for gene therapy have recently been reviewed in detail. J. C. Perales et al., An Evaluation of Receptor-Mediated Gene Transfer Using Synthetic DNA-Ligand Complexes. 226 Eur. J. Biochem., 255-266 (1994).
Cationic polymers such polyamine and PLL can be used as a DNA condensate. However, the use of cationic polymers alone as gene delivery carriers has several disadvantages. First, transfection efficiency is very low because they have no functional group except the amine group used in charge-neutralization. Also, due to the negative charges of the DNA phosphate backbone, an increase in the degree of charge neutralization of the DNA often results in extensive condensation and the separation of the DNA phase in the form of insoluble compact structures. Nucleoprotein Complexes: Studies on the Interaction of Cationic Homopolypeptides with DNA. 24 J. Mol. Biol., 157-176 (1967).
The asialoglycoprotein receptor mediates internalization of proteins bearing galactose-terminated oligosaccaride moieties into hepatocytes in liver. Therefore, the combination of a polymeric gene carrier with ligands such as galactose(Gal), lactose(Lac), and apoprotein E has been studied to deliver genes into hepatocytes. Although ligands directly conjugated to PEI with 5% galactose gave high transfection rates in hepatocyte-derived cell lines, they were unable to prevent aggregation under physiological saline conditions. Researchers have tried to combine pegylation and galactosylation for synthesis of new polymeric gene carriers using PEI. However, these synthesis methods seem too complex and these products show low gene expression due to less than optimal galactosylated content. P. Erbacher, T. Bettinger, P. Belguise-Valladier, S. Zou, J. L. Coll, J. P. Behr, J. S. Remy, Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI). J. Gene Med. 1 (1999) 210-222.
In view of the foregoing, it will be appreciated that providing a targeted composition of gene therapy and a method making thereof would be a significant advancement in the art.
The present invention provides a composition that efficiently mediates DNA delivery into a target cell. The present invention further provides a biocompatible composition for efficient DNA delivery which causes non-cytotoxic transfection into a target cell.
The present invention relates to compositions containing a cationic polymer(CP), i.e. a polyamine(PA), having a certain percentage of its cationic functional groups grafted with poly(ethylene glycol)(PEG) which is in turn covalently bound to a targeting moiety(TM). One embodiment of the present invention relates to Gal-PEG-PEI for gene delivery to hepatocytes. Furthermore, this composition mediates transfection of DNA or gene moieties into human cells. This transfection method and composition accomplish these goals while presenting minimal cell toxicity and significantly increased transfection efficiency.
Accordingly, the TM-PEG-CP conjugates of the present invention were synthesized by coupling the TM to an end of a PEG chain and covalently attaching or grafting the other end of the CP thus formed TM-PEG-CP. Particularly, the method comprising obtaining a TM derivative having an amine group and PEG containing a xcex-vinyl sulfone(VS) at one terminal and xcfx89-N-hydroxysuccinimidyl esters(NHS) at the other terminal, reacting the amine group of the TM with the VS group of the VS-PEG-NHS to obtain VS-PEG-TM; and then reacting the VS group of the VS-PEG-TM with the amine groups of the PA at a pH value of 4 to 9, preferably 6-9. The cationic polymer is preferably a polyamine(PA) such as polyalkylenimine(PAI) or PEI, and the targeting moiety is preferably lactose or galactose. The ratio of TM-PEG and polyamine(PA) can be adjusted by changing the ratio of reaction concentrations. The synthesized carrier, namely TM-PEG-PA, wherein 0.1 to 10 mole percent of the cationic groups on the CP are conjugated with TM-PEG with the rest of the cationic groups remaining unsubstituted, is capable of forming a stable and soluble complex with a nucleic acid, which in turn is capable of efficient transfection. The TM-PEG moiety grafted to a CP resulted in better solubility and reduced cytotoxicity of the nucleic acid/carrier complex as compared to CP alone. Compared with a CP without TM-PEG, the TM-PEG grafted PEI of this invention provides for high solubility of the complexes formed with DNA in physiological serum and cell culture medium, and prevents the precipitation and aggregation of the complexes formed, and thus is capable of being administrated in vivo at very high doses. The gene transfection efficiency and cytotoxicity of the TM-PEG-PLL system were investigated and compared to those of DNA complexed with a CP, such as PEI alone. HepG2 cells, as model cell lines of hepatocytes, were transfected specifically with a pDNA/Gal-PEG-PEI complex, indicating that galactose serves as a targeting moiety for hepatocytes. The TM-PEG-PEI of this invention also decreases the proteolytic degradation of DNA in the circulatory system and in cells, and enhances the uptake of the DNA/polymer complexes thus improving transfection efficiency. In addition, PEG also functions as a linker connecting the PEI backbone and the targeting moiety, thus increasing the targeting efficiency of DNA delivery to the target cells.